Neuroblastoma, the most common extracranial solid tumor of childhood, is a heterogeneous tumor. In some patients, the tumor can go into spontaneous regression and disappear whereas other patients have rapidly growing tumors with a poor prognosis. The overall long-term survival rate in patients with high-risk neuroblastoma is less than 30%, indicating the need for new treatment strategies. Angiogenesis inhibition hampers the formation of new blood vessels, thereby limiting the tumors’ metabolic exchange. Neuroblastoma is rapidly growing and high tumor angiogenesis has been associated with poor outcome. Therefore, the aim of this thesis was to investigate the effect of novel treatment modalities for angiogenesis inhibition on high-risk neuroblastoma xenografts. For that purpose, we used subcutaneous mouse models and characterized orthotopic mouse models for high-risk neuroblastoma. We found that xenotransplantation of neuroblastoma cells into the adrenal gland of SCID and SCID beige mice resulted in orthotopic tumors resembling clinical neuroblastoma in respect to tumor site, growth and spread. Using contrast-enhanced ultrasound, we observed that the receptor tyrosine kinase inhibitor SU11248 reduced orthotopic neuroblastoma growth and spread by reducing tumor angiogenesis. In subcutaneous xenografts for high-risk neuroblastoma, valuable for studies requiring continuous assessment of tumor volume, we demonstrated that immune-neutralizing VEGF with the anti-VEGF antibody bevacizumab significantly reduced neuroblastoma growth. Finally, we found that formulations of the chemotherapeutic drug GMX1778 inhibited angiogenesis and induced tumor regression in a dose dependent manner without host toxicity. We showed that relapsing tumors remained responsive to GMX-therapy without accelerated growth or induced drug resistance. In conclusion, SU11248, bevacizumab, and formulations of the active compound GMX1778 may become useful for treating high-risk neuroblastoma.

Background: Neuroblastoma (NB) is a pediatric tumor in infants and young children. The survival rate is only around 50 percent for high-risk NB despite advanced and intense multi-modal therapy. Current research aims to find new effective treatment additional to modern therapy to improve prognosis of high-risk NB in children. As such, SU11248 may be a valuable approach for improving treatment and survival as growth factors have crucial roles in tumor growth, angiogenesis, and metastasis. Aim: The aim of this investigation was to examine tissues from SU11248 treated and nontreated tumor-bearing animals on the abundance of tumor-associated macrophages (TAMs) in metastases found on vital organs. Our hypothesis is that if SU11248 could cause “deactivation” or “silencing” of the stroma of metastases particularly by acting on stromal immune cells such as TAMs. Methods: Paraffin-embedded metastases developed in an orthotopic xenograft model in beige SCID mice were stained with a monoclonal rat anti-mouse antibody as a marker of TAMs. Morphological analysis of tissue slides, and macrophage quantification was performed using a microscope. Statistical analysis was achieved using an unpaired two tailed t-test.  Results: Macrophages were stained nicely, but the number of macrophages in the metastases were not statistically different between the vehicle treated controls and SU11248 treated metastases. Conclusion: In patients with high-risk NB, SU11248 may be a useful therapeutic supplement. We believe that further research into mechanisms that target critical factors for angiogenesis and metastasis in NB, such as TAMs, is an important step toward improving patient outcomes in high-risk NB.

Background: Neuroblastoma is the most common extracranial solid tumor in children. The survival rate in high-risk neuroblastoma is less than 50 % despite intensive multimodal therapy, and there is thus an immense need for new treatment options. In a previous preclinical study conducted at Uppsala University, treatment with sunitinib was found to inhibit tumor growth and angiogenesis in an orthotopic model of high-risk neuroblastoma.  Aim: The present study aimed to further explore the effect of sunitinib on tumor stroma, focusing on whether it was possible to detect and quantify tumor-associated neutrophils (TANs) in tumor sections from the above-mentioned study using immunohistochemistry (IHC). Methods: Tissue sections from formalin-fixated paraffin-embedded tumors were stained with anti-Ly-6G/Ly-6C, anti-ITGAM, or hematoxylin-eosin, and the number of granulocytes was quantified manually using a light microscope. An independent samples two-tailed t-test was used for statistical analysis. Results: The average number of granulocytes increased by 40 % in animals treated with sunitinib compared to control animals (p = 0.003) in hematoxylin-eosin stained tumor sections of orthotopic neuroblastomas. The results from the staining with anti-Ly-6G/Ly-6C or anti-ITGAM, on the other hand, were impossible to quantify due to the high background staining despite the concentration of antibody used. Conclusion: In conclusion, this report indicates that the density of TANs in an orthotopic murine neuroblastoma model is increased by treatment with sunitinib. However, to confirm this result, the study should be repeated once a reliable IHC method for the detection of TANs has been developed.